A substantial body of literature has been developed concerning techniques that employ binding reactions, e.g., antigen-antibody reactions, nucleic acid hybridization and receptor-ligand reactions, for the sensitive measurement of analytes of interest in samples. The high degree of specificity in many biochemical binding systems has led to many assay methods and systems of value in a variety of markets including basic research, human and veterinary diagnostics, environmental monitoring and industrial testing. The presence of an analyte of interest may be measured by directly measuring the participation of the analyte in a binding reaction. In some approaches, this participation may be indicated through the measurement of an observable label attached to one or more of the binding materials.
One of the challenges with immunoassay measurements is the potential for cross-reactivity among antibodies and analytes. Cross-reactivity can lead to higher backgrounds, reduced sensitivity, and influence the detection of one analyte in a plurality. In a multiplexed immunoassay with N capture antibodies and N detection antibodies, there are N×N potential antibody interactions (including non-specific binding interactions). Therefore, in a 25-plex assay, there are 625 possible interactions. While antibodies are typically highly specific for a particular analyte, undesired interactions are common and have to be screened out when formulating a multiplexed assay panel. Finding antibodies that work well in a multiplexed format becomes increasingly challenging as the degree of multiplexing is increased.
In a multiplexed sandwich immunoassay format, the analyte specificity is provided by the capture antibody. As shown in FIG. 1(a), if the capture antibody (bound to a surface) and labeled detection antibody each bind to analyte A, A is detected in the assay. However, as shown in FIGS. 1(b)-(d), undesired interactions between capture and detection antibodies (1(b)), capture antibody and analyte B (1(c)), or capture and detection antibodies and an additional antibody present in the sample (1(d)) can result. If a capture antibody cross-reacts with another species in the sample, e.g., as shown in FIG. 1(b)-(d), the resulting signal can be misinterpreted as the presence of the target analyte, yielding a false positive result.
In addition, complex matrices, such as human serum/plasma and cell lysates, may contain molecules or molecular complexes that will crosslink capture antibodies to detection antibodies. This is particularly problematic as the effect is unpredictable (unlike direct antibody-antibody cross reactivity), and it can lead to falsely elevated measurements of a particular analyte. One example of a class of matrix-mediated cross-reactivity is human anti-mouse antibodies (knows as a HAMA effect), or other human anti-animal antibodies. When anti-mouse antibodies are present in human serum, they can bind to mouse-derived antibodies that are typically used in immunoassays, and potentially form a bridge between capture and detection antibodies, falsely mimicking the presence of an analyte. While this problem is relevant in single analyte immunoassays, it is exacerbated by a factor of N2 in multiplexed immunoassays where N detection antibodies can be bridged to N capture antibodies.